therapeutic and preventative anti-inflammatory benefits of … · 2014-07-09 · cumin is...

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For citation purposes: Alhagamhmad MH, Lemberg DA, Day AS, Leach ST. Therapeutic and preventative anti-inflammatory benefits of curcumin in vitro. OA Inflammation 2013 Oct 01;1(2):11. Co

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Therapeutic and preventative anti-inflammatory benefits of curcumin in vitro

MH Alhagamhmad1, DA Lemberg1,2, AS Day1,3, ST Leach1*

AbstractIntroductionIn the setting of inflammatory bowel disease, curcumin supplementation to prevent inflammatory flares has shown promise. However, the out-come of curcumin exposure, when there is existing inflammation, is not clear. The aim of this study was to compare the anti-inflammatory properties of curcumin when added at differing times to an inflammatory stimulus, in an established in vitro intestinal epithelial cell model of intestinal inflammation.Materials and MethodsHT-29 and INT407 cells were incu-bated with a range of concentrations of curcumin prior to, or at the same time as, the addition of tumour ne-crosis factor-α. Following incubation, cell viability, interleukin-8 levels and cytoplasmic inhibitor of kappa B (IκB) were assessed.ResultsHigh concentrations of curcumin reduced epithelial cell viability. At lower concentrations, curcumin had no effect on cell viability; however, curcumin (with or without pre–incu-bation) reduced interleukin–8 levels and inhibited the phosphorylation and degradation of IκB.Conclusion Curcumin can reduce interleukin-8 and IκB response to tumour necro-

sis factor–α in an in vitro model of intestinal inflammation. This re-sponse is not dependent on curcumin pre-incubation, but does depend on curcumin concentration. These find-ings support the role of curcumin as an anti-inflammatory therapy and suggest it may be effective in both reducing existing inflammation and preventing inflammatory relapse. Further research is needed to assess optimal in vivo curcumin dosing.

IntroductionTurmeric (the common name for Curcuma longa) is an Indian spice that belongs to the ginger family1. In ancient times, turmeric powder was utilised as a traditional and natural remedy for various health condi-tions, such as joint pain, ulcers, liver disease, wounds and skin diseases2. The active ingredient of turmeric is curcumin with chemical name diferuloylmethane3. Curcumin exhib-its anti-microbial, anti-inflammatory, anti-oxidant and anti-neoplastic properties4, and has been extensively investigated for its proposed benefits in managing chronic inflammatory conditions. In inflammatory bowel disease (IBD), curcumin is proposed as an attractive alternative therapy to the conventional medicines5,6. Cur-cumin is considered to be safe and inexpensive7 and emerging evidence from pre-clinical studies indicates the potential benefits of curcumin supplementation in IBD8–11.

Clinical trials of curcumin have in-cluded a pilot study involving 5 pa-tients with chronic ulcerative proctitis and 5 patients with Crohn’s disease (CD)12. Curcumin was administered for 2 months to patients with proc-titis and for 3 months to the patients

with CD.All patients with proctitis experienced symptom relief, which was consistent with a significant re-duction in inflammatory indices; in addition, four out of five patients were able to reduce concomitant medica-tions. Four of the CD patients who completed the study showed a sig-nificant drop in disease activity and erythrocyte sedimentation rate. Fur-ther, in a randomzsed, double-blind, multicentre trial involving 89 pa-tients with quiescent ulcerative co-liti), 45 patients received curcumin plus sulphasalazine or mesalamine, and 44 patients received placebo plus sulphasalazine or mesalamine for 6 months13. At the completion of the study, relapse rates were 4.65% in the curcumin-treated group compared to 20.51% in the placebo group. These preliminary studies appear promising and suggest that curcumin co-admin-istration with conventional medica-tions may be a viable therapeutic ap-proach. In addition to these human trials, promising results are also ob-served when curcumin is adminis-tered in experimental murine colitis models of IBD9,10,14–18. In these stud-ies, curcumin-treated mice exhibited symptomatic relief, improved surviv-al rates and decreased inflammatory indices. Addition of curcumin to mice diets resulted in a significant amelio-ration in the histological intestinal in-jury seen in the inflamed mucosa and lower mucosal cytokine levels19.

However, the role of curcumin in the management of active inflamma-tion is less clear. The aim of this study was to investigate whether timing of exposure to curcumin and/or cur-cumin concentration influenced the inflammatory response in an in vitro model of intestinal inflammation.

* Corresponding author Email: [email protected] School of Women’s and Children’s Health,

University of New South Wales Sydney, NSW, Australia

2 Department of Gastroenterology, Sydney Children’s Hospital, Randwick, Sydney, NSW, Australia

3 Paediatric Gastroenterology, Christchurch Hospital, Christchurch, New Zealand

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(Novex-Invitrogen, Victoria, Austral-ia). Briefly, following experiments, cell culture supernatants were col-lected and tested in duplicate. Sam-ples were added to 96-well microtitre plates (MaxisorpNunc, Victoria, Australia) coated with monoclonal IL-8 antibody and detected with bi-otinylated secondary antibody and streptavidin-horseradish peroxidase (HRP) conjugate. After development of the colourimetric reaction us-ing tetramethylbenzidine substrate (Thermo-Fischer Scientific, Victoria, Australia), the reaction was stopped with the addition of 1.8 N H2SO4. Ab-sorbance was measured at 450 nm by a Micro-plate reader (Bio-Rad). Ab-sorbance readings were then convert-ed to picograms per millilitre based on standard curves obtained with the recombinant cytokine (lower detec-tion limit of assay was 31.5 pg/ml).

Western blot analysis of IκBHT-29 cells were seeded in a 6-well plate at a concentration of 106 cells per well and grown for 5 days be-fore experiments. For some experi-ments, cells were treated with 50 µM curcumin for 1 hour before or at the same time during the administration of 100 ng/ml TNF-α. Cells were then incubated with TNF-α for 5, 15 and 30 minutes. Proteins were extracted from cells using radioimmunoprecip-itation assay buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% so-dium deoxycholate, 0.1% sodium do-decyl sulphate, 50 mM Tris, pH 8.0) containing 10 mM NaF and a mix-ture of protease inhibitors (2 µg/ml Aprotinin, 10 µg/ml Leupeptin and 1 µg/ml Pepstatin A). Cell lysates were centrifuged at 12,000 g for 10 minutes, and supernatants were collected. The total protein concen-tration of the cell lysates was meas-ured by the bicinchoninic acid method (Micro BCA protein assay reagent kit; Pierce Biotechnology-Thermo-Fischer Scientific). Equal amount of protein (40 µg) for all samples was loaded and run onto Tris-Glycine NB

of 1 mL of cells in suspension was transferred to a separate tube and centrifuged at 1200g for 5 minutes. Supernatant was discarded and the pellet containing the cells was resus-pended in warm media gently. Equal volumes of cells (25 µl) and trypan blue (Sigma-Aldrich) (25 µl) were mixed and cell viability counts were conducted using a haemocytometer and a light microscope. The viability was expressed as the percentage of unstained cells (viable) among the total cells.

Assessment of cell viability by MTT colourimetric assay kitsThe assay was conducted according to the manufacture’s instructions (Sigma-Aldrich). In brief, after 24 hours of incubation with curcumin, culture media was replaced with 1 ml per well phenol red-free McCoy´s 5A media (Banksia Scientific Compa-ny, QLD, Australia). One hundred mi-crolitres of MTT (3-[4,5-Dimethylth-iazol-2-yl]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue) solution (5 mg/ml) was added to each well of cells and incubated further for 4 hours at 37°C with 5% CO2. In meta-bolically active cells, the MTT was converted to an insoluble purple formazan by cleavage of the tetra-zolium ring by mitochondrial dehy-drogenase enzymes. Following the incubation, the dye was solubilised in DMSO and 200 µl of the mixture was transferred to a well of a 96-well plate. Well absorbance was read at a wavelength of 570 nM by a Micro-plate reader (Bio-Rad, NSW, Australia). The amount of converted dye, representing metabolic activity, was determined by comparing to a standard curve.

Enzyme-linked immunosorbent assay to measure interleukin-8The concentration of interleukin (IL)-8 in culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) kit, according to manufacturer’s protocol

Materials and MethodsThe protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed.

Cell culture and induction of inflammationHT-29 cells (ATCC HTB-38) were seeded in 24-well plates (Bec-ton Dickinson, NSW, Australia) at a concentration of 5×105 cells per well and maintained in McCoy’s 5A medium (Gibco®, Invitrogen, Vic-toria, Australia) containing 10% foetal bovine serum (FBS: Gibco®, In-vitrogen) and 100 U/mL penicillin/streptomycin (Gibco®, Invitrogen). INT407 (HeLa derivative) cells were also grown in 24-well plates and maintained in Basal Medium Eagle (Gibco® Invitrogen) containing 10% FBS and 100 U/mL penicillin/strep-tomycin. Cells were incubated at 37°C with 5% CO2 with media changed eve-ry alternative day. Experiments were conducted after 5 days of incubation or if 90% confluence was reached. In-flammation was induced by exposing cells to 50 ng/ml tumour necrosis fac-tor-alpha (TNF-α; Gibco®,-Invitrogen) and incubating HT-29 cells for 6 hours or INT407 cells for 24 hours.

Curcumin treatment protocolCurcumin was first dissolved in dime-thyl sulpfoxide (DMSO: Sigma-Aldrich, NSW, Australia), then mixed with a culture medium to give a final concen-tration of 10, 25, 50, 60, 75 or 100 µM curcumin with a final DMSO concen-tration of 0.1% v/v in culture media.

Assessment of cell viability by trypan blue exclusionFollowing experimentation, cells were washed twice with warm phosphate-buffered saline (Gibco®, Invitrogen), then incubated with trypsin- Ethylenediaminetetraacetic acid (EDTA) (Gibco®, Invitrogen) 0.5 mL per well for 15 minutes. Warm FBS (0.5 mL per well) was then add-ed to inhibit trypsin. A total volume

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10% gel (NuSep Ltd, NSW, Australia) for 1 hour. Proteins were then trans-ferred onto PVDF nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad Co), and then blocked with 5% bovine se-rum albumin in Tris-buffered saline solution with 0.1% polysorbate 20 (Tween 20; Sigma-Aldrich). Separate membranes were probed with the primary antibodies rabbit anti-IκB and anti-phosphorylated IκB (1:1000 dilution) for overnight incubation at 4°C (Abcam, Cambridge, UK); and total protein levels were assessed with anti-β-actin antibodies (Ab-cam, 1:1000 dilution). Subsequently, membranes were incubated with the secondary antibody goat anti-rabbit IgG (Bio-Rad, 1:25000 dilution) con-jugated with horseradish peroxidase (HRP) (Bio-Rad) for 1 hour at room temperature before bands were de-tected by chemiluminescent detec-tion method using the Immun-Star HRP Chemiluminescent Substrate Kit (Bio-Rad) and visualised by GelDoc (Bio-Rad).

Statistical analysisGraphPad Prism software (version 6.0 for windows; GraphPad Software, San Diego, CA, USA) was used for statisti-cal analysis. Results are presented as mean standard errors of mean (SEM). One-way ANOVA test with a Ficher’s least significance post-test was used for analysis. A p-value of < 0.05 was considered as statistically significant.

Results Curcumin reduces epithelial cell viability at high concentrationsHT-29 and INT407 cells were treated with increasing concentrations of curcumin (0, 10, 25, 50, 75 or 100 µM) in DMSO for 24 hours with cell viabil-ity measured. Additional DMSO only controls (cells exposed to 50 ng/ml TNF-α and DMSO without curcumin) were included. The final concentra-tion of DMSO for all experiments remained constant at 0.1% v/v, which did not have an effect on cell

Figure 1: Cell viability of HT29 and INT407 cells exposed to curcumin. HT29 (A) and INT407 (B) cells were exposed to curcumin at 0, 10, 25, 50, 75 and 100 µM dissolved in DMSO (0.1% v/v in culture media) and in-cubated for 24 hours. Cell viability was measured by trypan blue exclu-sin (*p < 0.05 vs. control group).

viability (Figure 1). Increasing con-centrations of curcumin, (up to 50 µM) had no significant effect on cell via-bility, with viability remaining above 90% for both the cell lines (p > 0.05) (Figure 1). However, curcumin con-centrations of 75 µM and above sig-nificantly decreased cell viability (p < 0.05 for both 75 and 100 µM curcumin) (Figure 1).

Cell viability results were further supported by the MTT assay experi-ments that measured the mitochon-drial dehydrogenase enzyme activity of the viable cells. HT-29 cells were treated with increasing concentra-tions of curcumin dissolved in DMSO (0, 10, 25, 50, 60, 75 and 100 µM) for 24 hours with cell activity measured. The final concentration of DMSO for all experiments including 0 µM curcumin treatment group (DMSO-control group) remained constant at 0.1% v/v. Curcumin, up to 50 µM concentrations, showed an increase in the mitochondrial dehydrogenase enzyme activity, while those treated with 60 µM or higher concentration of curcumin exhibited a significant drop in the activity as compared to the control group (p < 0.05 for 60, 75 and 100 µM treated groups) (Figure 2). Therefore, for further experimenta-tion, curcumin concentrations of up to 50 µM were used.

Curcumin inhibits IL-8 production from TNF-α-exposed HT-29 and INT407 cellsCurcumin exhibited a strong anti-inflammatory effect in response to TNF-α exposure of intestinal epithe-lial cells. In HT-29 cells, curcumin at low concentration (10 µM) in all the indicated pre-incubation time points (24, 6, 1 and 0 h) had no sig-nificant effection ameliorating IL-8 production from the cultured cells (p > 0.05 in all treated groups vs positive control group) (Figure 3A). However, at higher concentrations (25 and 50 µM), curcumin did signifi-cantly attenuatd IL-8 levels in a dose-dependent fashion. Cell exposed to

25 µM curcumin showed significant reductions in IL-8 levels compared to the positive control (p < 0.01) (Figure 3B). Interestingly, IL-8 levels were not significantly different be-tween cells with varying curcumin pre-incubation time (p > 0.05 for all comparisons at 24, 6, 1, and 0 hours) (Figure 3B), but by increasing cur-cumin concentration to 50 µM, the strongest anti-inflammatory re-sponse was observed with maximal reduction in IL-8 levels (p < 0.001 for all comparisons to positive controls;

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each time point following TNF-α ex-posure, which was consistent with less phosphorylated IκB detected (Figure 5). There was no difference in the IκB response whether or not cells were pre-incubated with cur-cumin prior to TNF-α exposure.

DiscussionThese in vitro investigations have demonstrated that curcumin can prevent TNF-α-mediated production of IL-8 in human intestinal epithe-lial cells. Further, inhibition of IL-8 was comparable whether cells were pre-incubation with curcumin or if cells were exposed to curcumin at the same time as TNF-α administra-tion. These results also indicatd that curcumin impaired the degrada-tion of IκB and thereby modulated the nuclear factor (NF)-κB signal transduction pathway.

In numerous experimental murine models of colitis, there is evidence that curcumin has potential therapeutic and preventive benefits that may be applicable in the treatment of IBD8,14,19. Previously, using the human intestinal cell line (HCT116, HT-29 and CaCO2), it was shown that curcumin pre-treatment inhibited and modulated gene expression of IL-8 in response to inflammatory cytokine exposure20,21. However, it was not investigated whether curcumin had a similar effect if given simultaneously with inflam-matory inducers. The results present-ed in this paper indicate that curcumin pre-incubation had no measureable additional benefit compared to when curcumin was given with the inflam-matory stimulus. However, curcumin concentrations did significantly affect IL-8 suppression. This finding indi-cates that curcumin concentration, bud not the timing of supplementa-tion, likely determines the efficacy of curcumin in reducing inflammation.

Curcumin inhibits the activity of cyclooxygenase, lipoxygenase and inducible nitric oxide synthetase enzymes; thereby reducing pro-in-flammatory cytokine production and

the curcumin concentration dose did affect IL-8 level (Figure 4D). DMSO alone at 0.1% v/v had no measurable effect on IL-8 production in either of the two cells lines (Figures 3 and 4).

Curcumin therapy suppresses IκB degradation in intestinal epithelial cells There is a rapid degradation of IκB when HT-29 cells are exposed to TNF-α. IκB is detectable by Western blot in unstimulated cells, but is un-detectable 5 and 15 minutes follow-ing TNF-α exposure (Figure 5). IκB can be detected again 30 minutes af-ter TNF-α exposure (Figure 5). These observations are consistent with the appearance of phosphorylated IκB at 5 minutes following TNF-α exposure, which indicates that IκB is being phosphorylated and subse-quently degraded by the proteasome activity (Figure 5). In cells exposed to curcumin, IκB can still be detected at

Figure 3C). Moreover, again timing of curcumin pre-incubation did not alter the anti-inflammatory response for cells exposed to 50 µM curcumin (p > 0.05 for all comparisons; Figure 3C). The dose-dependent curcumin response is clearly seen with no pre-incubation, where increase in the concentration of given curcumin re-sults in further reduction in IL-8 level (Figure 3D).

In INT407 cells, low concentra-tions of curcumin (10 and 25 µM) had no suppressing effect on IL-8 production; conversely, there was a rise in the IL-8 levels (Figures 4A and 4B). However, curcumin of 50 µM significantly reduced IL-8 pro-duction in response to inflammatory stimulus (p < 0.001 for all compari-sons compared to positive control; Figure 4C). Like in HT-29 cells, tim-ing of curcumin exposure dose did not influence this response (p > 0.05 for all comparisons; Figure 4C), but

Figure 2: Cellular activity assay of curcumin-treated cells. HT29 cells were ex-posed to curcumin at 0, 10, 25, 50, 60, 75 and 100 µM dissolved in DMSO (0.1% v/v in culture media) and incubated for 24 hours. Media was replaced with phe-nol red-free media and MTT substrate, incubated further and absorbance was read at 570nM (*p < 0.01;**p < 0.001 vs. 0 µM treated group).

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indicated that curcumin inhibits IκB degradation whether given prior to or at the same time as an inflamma-tion inducer. This suggests that cur-cumin gains rapid entry into the cells and become active and does not need to be metabolised to be activated.

It is important to note that, accord-ing to these experiments curcumin may have a narrow safe window. Cells treated with curcumin con-sentration greater than 50 µM had a significant drop in cell viability that was consistent with the reduction in the mitochondrial dehydrogenase enzyme activity. In contrast clinical trial has reported that patients were able to tolerate up to 8g of curcumin daily for 3 months wih no reported toxicity33. This apparent discrepancy is likely due te poor pharmacokinetic properties of curcumin, including very low bioavailabilit, poor absorp-tion and rapid metabolic elimina-tion34 when curcumin is delivered in the absence of a vehicle. Another study has found that the highest achieved peak serum concentration of curcumin in the peripheral blood was only 139 nM measured 4 hours after a single oral dose of 12 g cur-cumin35. Further, limited transepithe-lial flux and rapid metabolism in the gut epithelium are likely to impair curcumin exerting its beneficial ef-fect locally on gut epithelium36. In our in vitro experiments, curcumin was delivered via DMSO, which would en-hance delivery to the cell cytoplasm. Thus, this may explain why curcumin has shown better outcomes in both in vitro and ex- vivo settings than in clinical trials and may also explain the lack of toxicity despite high doses. Therefore, to improve clinical out-comes and to fully utiizse the prop-erties of this agent, future clinical application of curcumin may ben-efit from further investigation of cur-cumin solubility and targetd delivery of curcumin to the gut. However, im-proving delivery in vivo must also be done cautiously and with concurrent investigation of cell toxicity.

Thus, activity of the NF-κB pathway is determined by the state of the IκB protein. In this papere we report that a phosphorylated IκB band was evident shortly after the addition of TNF-α, which was consistent with the initial drop in IκB. Curcumin, with or without pre-incubation, appeared to prevent the rapid and initial loss of IκB. The lack of IκB degradation in curcumin-treated cells indicates inhibition of NF-κB signal transduc-tione A previous report involving an intestinal epithelial cell line indicat-ed that curcumin treatment blocks IL-8 expression through inhibition of NF-κB pathway21. Of interest, in that study, curcumin was pre-incu-bated with cells prior to exposure to inflammatory mediators. However, in the present study results have

down-regulating mitogen activated and Janus kinases22–24. These effects are primarily a result of influencing the NF-κB signal transduction path-way21,25–27. The NF-κB/Rel family of transcription factors plays a role in regulating the expression of a large number of genes, particularly those determining immune functions and acute-phase reactions28. Inhibition of NF-κB is of particular importance and is considered a putative target for in-tervention in IBD29,30. In unstimulated cells, most of the Rel/NF-κB factors are sequestered in the cytoplasm as inactive complexes bh associatinn with a group of inhibitory proteins (IκB)31. Once phosphorylated, Iκl dissociates from the NF-κB complex and permits the subsequent trans-location of NF-κB to the nucleus32.

(a) (b)

(c) (d)Figure 3: Effect of curcumin on IL-8 production from HT29 cells in response to TNF-α stimulation. HT29 cells were grown to confluence and pre-treated with varying concentrations of curcumin (10 [A], 25 [B] and 50 [C] µM) for varying times (24, 6, 1 and 0 [D] h). TNF-α (50 ng/ml) was added at time 0 and incubated for a further 6 hours. Supernatants were collected and assayed for IL-8 by ELISA. [Analysis of data was conducted using one-way ANOVA test followed b Fischer’s least significanc(*p < 0.0;*p < 0.001; ns > 0.05 vs. positive control group)].

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(a) (b)

(c) (d)Figure 4: Effect of curcumin on IL-8 production from INT407 cell line in response to TNF-α stimulation. INT407 cells were grown to confluence and pre-treated with varying concentrations of curcumin (10 [A], 25 [B] and 50 [C] µM) for varying times (24, 6, 1 and 0 [D] h). TNF-α (50 ng/ml) was added at time 0 and incubated for a furthe 6 h. Supernatants were collected and assayed for IL-8 by ELISA. [Analysis of data was conducted using one-way ANOVA test followed by Fischer’s least significance (*p < 0.001; **p < 0.01, ns > 0.05 vs. positive control group)].

Figure 5: IκB response in HT29 cells. Confluent cells were treated with DMSO-dissolved curcumin either for a 1-hour pre-incubation or at the same time as TNF-α exposure. Following TNF-α (100 ng/ml) exposure, cells were incubated for 5, 15 and 30 minutes, then cell lysates were collected. IκB levels in lysates were analysed by Western blot technique using anti-IκB (37 KDa) and anti-phosphorylated IκB (40 KDa), and total protein levels were assessed with anti-β-actin antibodies (40 KDa). Curcumin treatments with or without pre-incubation block IκB phosphorylation and thereby its degradation in TNF-α-stimulated HT29 cells.

Limitations of this study are re-lated to the use of HT-29 and INT407 epithelial moolayers. HT-29 cells are tumour-derived colonic epithelial cells and INT407 cells are human em-bryo small intestinal epithelial cells. These cell lines provide an approxi-mation of the human gut epithelial surface, but may not precisely reflect normal human intestinal physiology in vivo. However, due to their criti-cal role in the gut mucosal immune response37, epithelial cell lines are very often used as an in vitro model to reflect in vivo event38–42. Intesti-nal epithelial cells act as a protective barrier against different invasive in-fectious agents, toxins and antigenic factors within the gut lumen43,44, and are also in contact with cytokines secreted by other cells in the intesti-nal mucosa45. In addition, intestinal epithelial cells secrete numerous cy-tokines involved in intestinal injury and damage46–48. Thus, the in vitro re-sults presented here can be of value when contemplating in vivo curcumin supplementation.

ConclusionIn summary, curcumin prevented and ameliorated TNF-α-induced in-flammation in two intestinal cell lines. This action was mediated through inhibition of IκB degrada-tion, and therefore the NF-κB signal transduction pathway is also blocked in a similar way as with or without pre-incubation. This study further adds support that curcumin has the potential to improve intestinal dis-eases associated with inflammation such as IBD. This study suggests that curcumin does not likely need to be metabolised and is active in its natural state. Therefore, curcumin supplementation may be useful to both prevent inflammatory relapse episodes and treat current inflam-matory flare. However, curcumin concentration and delivery method are likely to be essential for the suc-cessful therapeutic utilisation of cur-cumin, and definitive clinical studies

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are now required to establish the op-timal dose and delivery regimen to ensure efficacy and safety.

Abbreviations listCD, Crohn’s disease; DMSO, dimethyl sulphoxide; EDTA, Ethylenediami-netetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FBS, foetal bovine serum; HRP, horserad-ish peroxidase; IBD, inflammatory bowel disease; IκB, inhibitor of kap-pa B; NF-κB, nuclear factor-κB; SEM, standard errors of mean; TNF-a, tu-mour necrosis factor-alpha

AcknowledgementsThis work was supported by the Xstrata coal community partner-ship program. MHA is funded by the Libyan government and would like to acknowledge Libya for that support. All experiments were conducted in the Westfield Research Laboratories.

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